Global distribution of α/β hydrolase family macrolide esterases in Gram-positive bacteria

Abstract Macrolide antibiotics are vital for controlling infections in humans, animals, and agriculture, yet their effectiveness is increasingly compromised by antimicrobial resistance. Macrolide esterases (MLEs) are key mediators of macrolide resistance but have only been detected in Gram-negative bacteria, with no evidence in Gram-positive species. Here, we mined over 500,000 Gram-positive genomes and identified 8,707 candidate proteins. Six representative MLEs were functionally validated, conferring resistance to 16-membered macrolides and increasing minimum inhibitory concentrations up to 16-fold in Escherichia coli and 128-fold in Bacillus subtilis. Moreover, two exhibited broad-spectrum activity against all clinically and veterinary relevant 16-membered macrolides. Temporal analysis revealed that Gram-positive MLEs originated at least 2.7 million years ago, contrasting with their emergence in Gram-negative bacteria after the introduction of antibiotics. Genomic surveys further demonstrated the global distribution of MLE-carrying Gram-positive bacteria across 97 countries and diverse ecosystems, including clinical, food, agricultural, and natural environments. These findings highlight Gram-positive MLEs as an underrecognized risk and underscore the need for a One Health–oriented strategy to monitor, assess, and mitigate the spread of macrolide resistance across interconnected ecosystems.